BRAF mutation T1796A in thyroid cancers

ABSTRACT

The BRAF gene has been found to be activated by mutation in human cancers, predominantly in malignant melanoma. We tested 476 primary tumors, including 214 lung, 126 head and neck, 54 thyroid, 27 bladder, 38 cervical, and 17 prostate cancers, for the BRAF T1796A mutation by polymerase chain reaction (PCR)-restriction enzyme analysis of BRAF exon 15. In 24 (69%) of the 35 papillary thyroid carcinomas examined, we found a missense thymine (T)→adenine (A) transversion at nucleotide 1796 in the BRAF gene (T1796A). The T1796A mutation was detected in four lung cancers and in six head and neck cancers but not in bladder, cervical, or prostate cancers. Our data suggest that activating BRAF mutations may be an important event in the development of papillary thyroid cancer. Moreover, BRAF mutation reliably predicts a poor prognosis for papillary thyroid carcinomas.

This application claims priority to provisional U.S. Application Ser.No. 60/462,046, filed Apr. 14, 2003.

FIELD OF THE INVENTION

The invention relates to diagnostic, therapeutic, and prognostic methodsfor thyroid cancers.

BACKGROUND OF THE INVENTION

Raf kinase is a key component of the RAS→Raf→MEK→ERK/MAP kinasesignaling pathway, which plays a fundamental role in the regulation ofcell growth, division and proliferation, and, when constitutivelyactivated, causes tumorigenesis (19). Among several isoforms of Rafkinase, the B-type, or BRAF, is the strongest activator of thedownstream MAP kinase signaling (25). The BRAF gene is located onChromosome 7.

The RAF proteins are highly conserved serine/threonine protein kinasesthat have an important role in cell proliferation, differentiation, andprogrammed cell death (1). The RAF proteins activate mitogen-activatedprotein kinase kinase (MEK), which in turn activates themitogen-activated protein kinase (MAPK) pathway (2). Inappropriateand/or continuous activation of this pathway provides a potentpromitogenic force resulting in abnormal proliferation anddifferentiation in many human cancers (3). Davies et al. (4) reportedthat BRAF is frequently mutated in a variety of human tumors, especiallyin malignant melanoma and colon carcinoma. The most common reportedmutation was a missense thymine (T)→adenine (A) transversion atnucleotide 1796 (T1796A; amino acid change in the BRAF protein isVal⁵⁹⁹→Glu⁵⁹⁹) observed in 80% of the malignant melanoma tumors.Functional analysis revealed that this transversion was the onlydetected mutation that caused constitutive activation of BRAF kinaseactivity, independent of RAS activation, by converting BRAF into adominant transforming protein (4).

Papillary thyroid cancer (PTC) is the most common thyroid cancer,accounting for about 80% of thyroid malignancies (20). Although PTC isusually indolent and curable with surgical thyroidectomy followed byradioiodine treatment, many patients do have recurrence and some becomeincurable and die (18); (15); (17); (24). Consequently, it is importantto undertake appropriate risk stratification and prognostic evaluationfor patients with PTC in order to provide optimal clinical management ofthe cancer. This is usually achieved based on evaluation of variousclinicopathologic risk factors, such as the age and gender of thepatient, the size of the tumor, and extrathyroidal invasion andmetastasis status (18); (15); (17); (24). With the demonstration of theoncogenic effect of the BRAF T1796A transversion mutation (4), it isconceivable that BRAF mutation plays an important tumorigenic role inPTC and may thus affect the clinicopathologic outcomes of these cancers.Indeed, it has recently been shown that BRAF mutation was associatedwith a higher prevalence of extrathyroidal invasion and advancedpathologic stage of PTC (27). In another recent analysis on PTC,however, no significant association of the BRAF mutation withextrathyroidal invasion was demonstrated although a marginallysignificant association of the BRAF mutation with advanced pathologicstage was observed (26). There is a need in the art for improved and/oradditional means for detecting, diagnosing, categorizing, treating, andpredicting outcomes for thyroid cancers.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment of the invention a method is provided fordistinguishing malignant from benign thyroid samples. The presence of aT→A transversion at nucleotide 1796 of BRAF according to SEQ ID NO: 1 isdetermined in a thyroid sample of a human. The presence of thetransversion indicates a malignant thyroid neoplasm and absence of thetransversion indicates a benign neoplasm or sample.

In a second embodiment a method for distinguishing malignant from benignthyroid samples is provided. The presence of a T→A transversion atnucleotide 1796 of BRAF according to SEQ ID NO: 1 is determined in ablood sample of a human. The presence of the transversion indicates amalignant thyroid neoplasm in the human and absence of the transversionindicates a benign neoplasm or no neoplasm.

In a third embodiment of the invention a method is provided fordetecting a mutation at nucleotide 1796 of BRAF. All or part of exon 15of BRAF from a test sample is amplified to form amplified products. Thepart of exon 15 comprises at least nucleotides 1792 to 1799 of BRAF. Theamplified products are digested with restriction endonuclease TspRI toform digested products. A mutation at nucleotide 1796 is identified ifthe digested products contain one fragment fewer than digested productsformed when using wild-type BRAF as a template for amplifying anddigesting; or one additional fragment compared to digested productsformed when using wild-type BRAF as a template for amplifying ordigesting.

In a fourth embodiment of the invention a method is provided fortreating a thyroid cancer patient. An effective amount of an inhibitorof BRAF serine/threonine kinase is administered to the patient.

In a fifth embodiment of the invention a method is provided for treatinga thyroid cancer patient. An effective amount of an inhibitor of theRas-Raf-MAPK pathway or the Raf/MEK/ERK signaling pathway isadministered to the patient.

These and other embodiments of the invention provide the art withadditional methods to successfully manage thyroid cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. TspRI restriction enzyme analysis (FIG. 1A) and exon 15 sequenceanalysis (FIG. 1B) of BRAF. FIG. 1A) Restriction pattern of the T1796Amutation. Lane M=mutant T299; lane WT=wild-type T486. FIG. 1B) ManualDNA sequence gel of exon 15 from papillary thyroid samples harboring theT1796A mutation (arrowhead). Lane 1=T569; lane 2=T203; lane 3=a thyroidadenomatous hyperplasia (T530) negative for the T1796A mutation; lane4=T228; lane 5=T171; and lane 6=melanoma cell line HTB72 that carries ahomozygous T1796A mutation. The sequence is to the right.

FIG. 2. Kaplan-Meier estimate of cancer recurrence-free probability inBRAF mutation-positive and -negative papillary thyroid cancers. Shortvertical lines indicate censored observations (months of follow-up forthose that have not had a recurrence). Log-rank chi-square=4.28,p=0.039.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present inventors that T1796A mutations of theBRAF gene are common in thyroid carcinomas and that they can be used todistinguish between benign and malignant carcinomas. Moreover, thepresence of the mutation bodes ill for a patient's survival and for therecurrence of the cancer.

The transversion mutation T1796A of the BRAF gene is known in the art.Any method of detecting the mutation may be used. These include, but arenot limited to, amplifying and sequencing the region of the genecontaining nucleotide 1796, amplifying and digesting with a restrictionendonuclease whose recognition or cleavage site is destroyed by themutation (such as TspRI), primer extension methods, and hybridization toallele specific oligonucleotides. Any method known in the art fordetecting point mutations can be used. Genomic DNA, mRNA, or protein ofthe BRAF gene may be assayed to determine the transversion mutation.

A variety of samples from the patient can be tested for the transversionmutation. These include tissue samples, cytological samples such as fineneedle aspirates, and blood. Blood samples include inter alia wholeblood, serum, and plasma. Sputum, saliva, lymph, tears can also betested. If a tissue sample is used it can have a follicular, apapillary, an indeterminate, or an undetermined morphology.

Determination of the presence or absence of the transversion in a samplecan be used to provide a diagnosis, to decide whether to performsurgery, and to decide what drugs and or radiation will be used. Drugswhich may be prescribed include chemotherapeutic agents, therapeuticantibodies, therapeutic anti-sense oligonucleotides or constructions,small interference RNAs, etc. A prognosis may constitute an estimatedlife expectancy, a prediction of recurrence, or a recommendation for anaggressive or non-aggressive therapeutic regimen. A prognosis may alsoinclude a recommendation of a schedule for monitoring for recurrence ormetastasis.

When using a restriction endonuclease assay to detect a transversion atnucleotide 1796, one typically amplifies a fragment of the BRAF genewhich comprises this nucleotide. The fragment will typically have asufficient number of nucleotides on either side of the transversion suchthat a change in size of a fragment containing the transversion will bereadily detectable. Moreover, a sufficient number of nucleotides will bepresent such that the full recognition and/or cleavage site is presentin the fragment. For example, when using the enzyme TspRI in an assay,at least nucleotides 1792 to 1799 will typically be present in thefragment. If a restriction enzyme cleavage and/or recognition site isdestroyed by a transversion mutation, then two fragments of a wild-typeallele will be joined. Thus from the mutant allele, there will be onefewer fragments. If both alleles in the sample are mutant, then therewill be one fewer fragment in the sample. If only one allele is mutantand one allele remains wild-type, then the sample will have anadditional fragment (the fused fragments) relative to a wild-typesample.

Thyroid cancers can be treated using any agent which will inhibit BRAFexpression or activity or an inhibitor of the Ras-Raf-MAPK orRaf/MEK/ERK pathways. Typically the presence of a BRAF transversionmutation will be detected prior to treatment. Suitable inhibitorsinclude antibodies that bind to BRAF kinase or other components of thepathway. The antibodies can, but need not, bind preferentially to BRAFrelative to other isoforms of RAF kinase. Antisense oligonucleotides canbe administered. The antisense oligonucleotides typically will havemodified chemical structures to enhance stability in the body. One suchmodified structure contains phosphorothioates in the phosphate backbone.Other modifications which reduce nuclease degradation and retainsusceptibility to RNase H can also be used. For example, 2′-O-methylnucleosides can be used, particularly on the 5′ and 3′ ends. Theoligonucleotides can be complementary to BRAF mRNA or to other mRNAencoding components of the pathway. Some such oligonucleotides which canbe used are ISIS 5132 and ISIS 13650. Oligonucleotides can becomplementary to various portions of the mRNA. The region surroundingthe start codon may be targeted, as can splice sites. Double strandedinhibitory RNA molecules can also be used. These are typically about20-26 bases in length or preferably 20-23 bases in length. The moleculespreferably contain 2-nucleotide 3′ overhangs. They can be complementaryto BRAF mRNA or to mRNA encoding other components of the pathway. Smallmolecule inhibitors of the kinase activity of BRAF or the pathways canalso be used. Such inhibitors include CI 1040 and BAY 43-9006.

Papillary and follicular thyroid carcinomas originate from thyroidfollicular epithelial cells. To date, oncogenic mutations in RAS andRET/PTC rearrangements have been observed in follicular thyroidcarcinoma and papillary thyroid carcinomas, respectively (5,6). RASmutations are common in follicular thyroid cancers, reaching 50% in somestudies, but are less common (5%-20%) in papillary thyroid tumors (5).Our observation of a high frequency of BRAF-activating mutations inpapillary thyroid carcinoma suggests that BRAF activation and, in turn,activation of the RAF/MEK/MAPK signaling pathway, is a common biologicmechanism in the development of human papillary thyroid carcinoma. Thisobservation is also consistent with the reported inverse associationbetween the presence of BRAF and RAS mutations in other cancer types(4,7,8). The relationship between BRAF T1796A mutation and RET/PTCrearrangements remains to be explored.

The importance of the RAS pathway in thyroid cancers is furthersuggested by the common presence of RET mutations in medullary thyroidtumors and their transforming effect through activation of theRAS/RAF/MEK pathway (9). Moreover, activation of the RAS/RAF/MEK/MAPKpathway is known to induce genomic instability in thyroid PCCL-3 cells(10), and inhibition of the MAPK pathway has led to decreased cellularproliferation of human thyroid cancer cell lines (11). Thus, activationat various points in the RAS/RAF/MEK/MAPK pathway is a key event in themost common type of malignant thyroid tumor. The high frequency of BRAFmutations in melanoma and papillary thyroid carcinoma suggests thatinhibition of BRAF activity by the newly developed RAF kinase inhibitors(12) may offer a new strategy in the treatment of these tumors. Ourresults have identified the BRAF T1796A mutation and the activation ofthe RAF/MEK/MAPK signaling pathway as a major mechanism in thedevelopment of primary papillary thyroid carcinoma.

PTC is the most common thyroid cancer, accounting for the vast majorityof thyroid malignancies. Although PTC is generally an indolent cancerand, with the current standard treatments, has an excellent long-termsurvival rate (18,15,17,24), many patients have recurrence and somebecome incurable and die. To reduce the morbidity and mortality furtherand improve the efficiency of the current management for PTC patients,it is important to conduct a proper risk stratification and prognosticevaluation in order to optimize the management for these patients. Thisis traditionally achieved based on clinicopathologic factors of thetumor and the patient that may affect the disease outcome. Large tumorsize, extrathyroidal invasion, distant metastasis, advanced pathologicalstage of the tumor, multifocality, older age of the patient at time ofdiagnosis, and male gender are all to some extent associated with apoorer prognosis (18,15,17,24). Neck lymph node metastasis, particularlywhen with extracapsular invasion, was shown to predict tumor recurrence(33). In general, though, the prognostic significance of lymph nodemetastasis in PTC patients may be related to the age of patient atdiagnosis (17). In young patients, neck lymph node involvement may notimpose significant risk for tumor progression and recurrence,particularly if the patient has properly received post-operativeradioiodine ablation therapy. In older patients, however, lymph nodeinvolvement is associated with increased risk of cancer progression andrecurrence. Among all these risk factors, extrathyroidal invasion is oneof the most reliable prognostic factors that predict a high probabilityof cancer progression and recurrence. Some of the other factors,however, do not seem to be consistently good prognostic factors in allstudies. An effective and reliable prognostic factor, such as a mutationbiomarker, could improve further the current risk and prognosticevaluation of PTC clinically.

The T1796A transversion BRAF mutation is the most common known geneticalteration in thyroid cancer and exclusively occurs in PTC(23,13,28,32,16,26,27,29). As this mutation was demonstrated to beoncogenic (4), we proposed that it played a significant role in thyroidtumorigenesis and in determining the phenotypic behaviors of PTC andtherefore might be a useful biomarker that can be added to the panel ofthe prognostic factors discussed above for the risk evaluation of PTCpatients. We therefore have examined the BRAF T1796A transversionmutation in PTC from a large group of patients and analyzed thecorrelation of this mutation with those clinicopathologic parametersthat are known to predict a poor prognosis of PTC. We found asignificant association of this mutation with tumor extrathyroidalinvasion, advanced pathologic stages, and neck lymph node metastasis,and, not surprisingly, also with a higher incidence of cancer recurrenceduring the clinical follow-up after thyroidectomy (Table 2). Thisassociation still existed even after a multivariate analysis with anadjustment for the potential confounding factors including patient ageand gender, tumor size and multifocality, and radioactive 1-131treatments (Table 4). Kaplan-Meier estimate of the effect of BRAFmutation on tumor recurrence clearly showed a poorer tumorrecurrence-free probability for BRAF mutation-positive PTC patients(FIG. 2), and, even after adjustment for the above-mentioned confoundingvariables, the relative risk for tumor recurrence associated with BRAFmutation was statistically significant. Moreover, when differentsubtypes of PTC were analyzed, we found that BRAF mutation occurred morefrequently in those subtypes of PTC (classic and tall cell variant) thatwere associated more often with extrathyroidal invasion, lymph nodemetastasis, and cancer recurrence (Table 3). Our study thereforesuggests that BRAF mutation plays a significant role in determining thephenotypic behaviors of PTC and is a genetic indicator of poor prognosisfor this type of thyroid cancer.

In a recent study on a series of PTC that was comprised mostly ofAmerican patients, a significant association of BRAF mutation withextrathyroidal invasion and advanced stages of the tumor was observed(27). In this study, unlike in the present one, no multivariate analysison each of these pathologic parameters for an adjustment of potentialconfounding factors was performed. Clinical follow-up data on thepatients were also not reported and therefore how BRAF mutation affectedthe cancer recurrence—an ultimate clinical outcome of the patients—wasnot clear. In addition, although a higher prevalence of lymph nodemetastasis was apparently associated with BRAF mutation in this study,it did not reach statistical significance, perhaps due to the need for aeven larger number of PTC samples. Interestingly, this previous studyshowed a significant association of BRAF mutation with older age of thepatients while ours failed to show so. A recent study on a largeJapanese series of PTC with a similar number of cases to ours onlyshowed a marginally significant association of BRAF mutation withadvanced tumor stage, but no significant association of BRAF mutationwith extrathyroidal invasion and lymph node metastasis was observed(26). However, BRAF mutation was associated with a higher incidence ofdistant metastasis in this study, although this large Japanese serieshas reported the lowest prevalence of BRAF mutation (29%) in PTC amongthe studies that have been reported so far. It is not clear if the roleof BRAF mutation varies in PTC of patients from different ethnicbackgrounds. The relationship of BRAF mutation with other knownrisk/prognostic factors apparently remains to be clarified in largerstudies.

In summary, our work with careful statistical analysis has expanded theprevious studies and demonstrated that BRAF mutation is associated witha number of pathologic features of PTC that are known to be predictorsfor a poor prognosis. We further showed that BRAF mutation wasassociated with a higher incidence of tumor recurrence, demonstratingthe usefulness of BRAF mutation as a novel prognostic genetic marker inthe risk and prognostic evaluation of patients with PTC. We suggest thatit may be a useful strategy to examine the BRAF mutation status in thethyroid tumor of patients with PTC to identify those with BRAF mutationso they can be optimally managed. In this context, preoperativeexamination of BRAF mutation on fine needle aspiration specimens, whichwas recently demonstrated to be a reliable and sensitive method todetect BRAF mutation (30), could provide valuable guidance in planningfor optimal thyroid surgery and subsequent vigilant clinical follow-upin patients with PTC.

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1

In this study, we investigated the frequency of BRAF T1796A mutation andfurther elucidated the importance of this mutation in various primaryhuman tumors.

We screened 476 primary tumors, including 214 lung, 126 head and neck,54 thyroid, 27 bladder, 38 cervical, and 17 prostate cancers for theBRAF T1796A mutation by polymerase chain reaction (PCR)-restrictionenzyme analysis. The samples were obtained from patients treated at TheJohns Hopkins Medical Institutions (Baltimore, Md.) and were collectedin our tissue bank. Written informed consent was obtained from eachpatient in accordance with the institutional review board at The JohnsHopkins Medical Institutions. PCR amplification of exon 15 followed bydigestion of the exon 15 products by the restriction endonuclease TspRIidentified the BRAF T1796A mutation. TspRI digestion of the PCR fragmentyielded three major bands at 125 base pairs (bp), 87 bp, and 12 bp inthe wild-type allele. The T1796A mutation abolished the restrictionsite, resulting in a prominent 212-bp band from the mutant allele andresidual bands from the normal allele (FIG. 1, A⇓). Reamplification ofBRAF exon 15 followed by direct manual sequencing of five samplesvalidated the results of the TspRI assay (FIG. 1, B⇓). As positivecontrols for the BRAF T1796A mutation, we used melanoma cell linesHTB71, HTB72, and A2058; for negative controls, we used cell lines ME180(cervical cancer) and HCT116 (colorectal carcinoma).

The BRAF T1796A mutation was identified in 24 (69%) of 35 papillarythyroid carcinomas (Table 1⇓), six (4.8%) of 126 head and neck cancers,and four (1.9%) of 214 lung cancers. Moreover, we analyzed nine commonthyroid cell lines (KAK1, KAT5, KAT7, KAT10, DRO, ARO, MRO 87-1,WRO-821, and C643) and found the same BRAF mutation in six (67%) of thenine lines. We also completely sequenced exons 11 and 15 in allT1796A-negative papillary thyroid cancers and in 10 T1796A-positivetumors but did not identify additional BRAF mutations. We did notidentify any mutations in bladder, cervical, and prostate primarytumors, and no mutation was identified in biopsy samples from 20patients with benign thyroid conditions (nodular goiter, follicularadenoma, atypical follicular adenoma, and adenomatous hyperplasia), 13patients with follicular thyroid carcinoma, three patients withmedullary thyroid carcinoma, and three patients with Hürthle cellcarcinoma.

Example 2

In the present study on a large series of thyroid cancer patients, weinvestigated the correlation of BRAF mutation with variousclinicopathologic characteristics of the tumor and, to take a furtherstep, we also examined the effect of BRAF mutation on cancer recurrenceover a long period of clinical follow-up. We found a significantassociation of BRAF mutation with extrathyroidal invasion and advancedpathologic stages of the tumor. In addition, we also observed asignificantly higher association of BRAF mutation with neck lymph nodemetastasis and cancer recurrence, demonstrating that BRAF mutation is anovel prognostic biomarker that predicts a poor prognosis for PTC.

Patients and Clinicopathologic Data Collection

Based on a protocol approved by the Institutional Review Board of theJohns Hopkins University School of Medicine and with appropriate patientconsent, we retrospectively reviewed the clinical records of 171patients who had thyroidectomy for thyroid tumors over the last 10 yearsat the Johns Hopkins Medical Institutions (including Johns HopkinsHospital and Johns Hopkins Bayview Medical Center) and whose thyroidtumor tissues were available for BRAF mutation analysis. Information onclinicopathologic characteristics of the tumor and clinical course ofeach patient (e.g., cancer recurrence and history of radioiodinetreatment), as specified in the section of Results, was collectedthrough this review. The histopathologic description, including thehistological diagnosis and tumor subtype classification, was made byseveral experienced pathologists at the Johns Hopkins MedicalInstitutions based on standard criteria. The tumors studied included 123PTC, 6 follicular thyroid cancers, 3 Hurthle cell thyroid cancers, 3medullary thyroid cancers, and 36 benign thyroid neoplasms. Because BRAFmutation was found exclusively in PTC, only the PTC patients wereanalyzed for the correlation between the clinicopathologiccharacteristics and BRAF mutation status of the tumor. The demographicinformation of these patients is shown in Table 2 of the Resultssection. All the PTC patients received total or near totalthyroidectomy. The clinical “follow-up” time for those patients who hadcancer recurrence was defined as the time period from the initialthyroid surgery to the first tumor recurrence. For those patients whodid not have cancer recurrence, the “follow-up” time was defined as thetime from the initial thyroid surgery to the most recent clinicalevaluation at the Johns Hopkins Medical Institutions. The follow-updurations for each group of patients are shown in Table 2.

Thyroid Tumor Tissues and DNA Isolation

Fresh frozen or paraffin-embedded PTC samples from patients weremicrodissected and DNA isolated as previously described (31). Briefly,after microdissection of the tissues, and after a 3-h treatment at 48°C. with xylene for tissues dissected from paraffin-embedded samples, thesamples were subjected to digestion with 1% SDS and 0.5 mg/ml proteinaseK at 48° C. for 48 h. To facilitate the digestion, a midintervaladdition of a spiking aliquot of concentrated SDS-proteinase K was addedto the sample tubes. DNA was then isolated from the digested tissues bystandard phenol-chloroform extraction and ethanol precipitationprocedures. In some cases, DNA isolated from fine needle biopsyspecimens was used instead.

Detection of BRAF Mutation

The BRAF T1796A mutation was analyzed on all the tumor samples in thepresent study, using genomic DNA by direct sequencing and a colorimetricmethod using the Mutector Kit (TrimGen, Baltimore, Md.) following themanufacturer's instructions. For direct DNA sequencing, exon 15 of theBRAF gene was amplified using the primers previously described (4):TCATAATGCTTGCTCTGATAGGA (SEQ ID NO: 2; forward) andGGCCAAAAATTTAATCAGTGGA (SEQ ID NO: 3; reverse). The PCR was performedusing a step-down protocol: 95° C. for 5 minutes×1 cycle; 95° C. for 1minute, 60° C. for 1 minute, and 72° C. for 1 minute, ×2 cycles; 95° C.for 1 min, 58° C. for 1 minute, and 72° C. for 1 minute×2 cycles; 95° C.for 1 minute, 56° C. for 1 minute, and 72° C. for 1 minute×40 cycles;with a final extension at 72° C. for 5 minutes. The PCR products weresubsequently subjected to Big Dye terminator Cycle Sequencing reactionand the sequence was read on an ABI PRISM 3100 Genetic Analyser (AppliedBiosystems) and the T1796A mutation was identified on the nucleotidesequence.

The calorimetric method for BRAF mutation was based on the technique ofshifted termination assay, which was demonstrated to have a 100%sensitivity and specificity for the detection of BRAF mutation (30).Briefly, in this assay, a specifically designed detection primerhybridizes to the target sequence of the BRAF gene with its 3′ terminusending just before the target base. The primer extends through thetarget base only if it is a T1796A transversion mutation. The extensionends at a termination base and multiple labeled nucleotides areincorporated through this process. The procedure was started with PCRamplification of exon 15 of the BRAF gene as described above, followedby hybridization of the PCR products to the specific primers attached tothe strips. Primer extension was achieved through a PCR reaction. Colordevelopment was performed through an enzymatic reaction and theintensity of the color was measured at a wave length of 405 nm. Thederails were as described recently (30).

Statistical Analysis

Categorical data were summarized using frequencies and percents.Distributions of the continuous variables were assessed, and all but ageat diagnosis were found to not be normally distributed. Therefore, thesedata were summarized with medians and interquartile ranges. Groupcomparisons of categorical variables were performed using the chi-squaretest or, for small cell sizes, Fisher's exact test. Non-parametricstatistics were used to compare the continuous variables. Comparisons oftwo groups were evaluated with the Wilcoxon rank sum test andcomparisons of three groups were done using the Kruskal-Wallis test.Multivariate logistic regression analyses were performed to assess theeffect of BRAF mutation on clinicopathologic outcomes of tumor stage,neck lymph node metastasis, extrathyroidal invasion, and recurrence ofthe tumor, adjusting for age at diagnosis, gender, multifocality andtumor size. The analysis of the effect of BRAF mutation on tumorrecurrence was also adjusted for 1-131 treatment. Product-limit survivalanalysis (22) and the log-rank test (21) were used to evaluate theeffect of BRAF mutation on cancer recurrence. Proportional hazardsregression analysis on tumor recurrence (14) with adjustment for thesame variables as the dichotomous outcome, was performed to examine therisk for cancer recurrence associated with BRAF mutation. Confidenceintervals (CI) were computed by standard methods. All reported p valuesare two-sided. Analysis was performed using SAS Version 8.0 software(SAS Institute, Cary, N.C.).

Results

Confirmation of the T1796A BRAF Mutation in PTC

Since the T1796A transversion mutation is the most common BRAF mutationin human cancers (4) and is the only BRAF mutation found in thyroidcancer (specifically, PTC) with a high prevalence in all the studiesreported so far (23); (13); (28); (32); (16); (26); (27); (29), weanalyzed this particular mutation in thyroid tumors in the present studyusing both direct DNA sequencing technique and the calorimetric assay.As summarized in Table 1, consistent with previous reports, we foundBRAF mutation only in PTC, but not in the follicular thyroid cancers,Hurthle cell thyroid cancers, medullary thyroid cancers, and benignthyroid neoplasms. TABLE 1 Prevalence of BRAF Mutation in VariousThyroid Tumors BRAF Mutation// Total % Papillary (overall)  54/123 44Classic 40/69 58 Papillary Follicular  4/44 9 Variant Tall Cell 10/10100 Variant Follicular Cancer 0/6 0 Hurthle Cell Cancer 0/3 0 MedullaryCancer 0/3 0 Benign Neoplasms  0/36 0Association of BRAF Mutation with High-Risk Pathologic Features of thePTC and a Higher Incidence of Cancer Recurrence

We performed a clinicopathologic correlation analysis on BRAF mutationin the PTC patients. As shown in Table 2, the overall analysis of the123 PTC patients revealed a significant association of BRAF mutationwith extrathyroidal invasion, neck lymph node metastasis, and moreadvanced pathologic stages of the tumor at the initial thyroidoperation. These three pathologic features are traditionally thought tobe associated with a high risk for poor prognosis of thyroid cancer.BRAF mutation was also associated with a significantly higher incidenceof cancer recurrence after thyroidectomy. Except for two cases ofdistant metastasis with persistent disease, all the recurrences werelocal in the neck. There was no significant association of BRAF mutationwith a particular gender or age of the patient and the size ormultifocality of the tumor. There was also no significant difference inpostoperative radioiodine-131 treatment in terms of the number and dosesbetween the BRAF mutation-positive and negative groups. TABLE 2Correlation between clinicopathologic characteristics and BRAF mutationstatus in patients with papillary thyroid cancer. Median (interquartilerange) or N (%). BRAF+ BRAF− P value N (total) 54 69 Age at diagnosis 45.5 (35-58)  46.0 (37-56) 0.89 Gender, male   16 (30%)   22 (32%) 0.79Tumor Size, cm  2.0 (1.3-3.0)*  2.4 (1.5-3.5)** 0.13 Extrathyroidal   22(41%)    7 (10%) <.0001 invasion Lymph node   28 (55%)***   14 (20%)<.0001 metastasis Tumor stage 0.049 I   17 (35%)   22 (32%) II   16(33%)   37 (54%) III   14 (29%)    8 (12%) IV    2 (4%)    2 (3%) TumorStage, III/IV   16 (33%)⁺   10 (14%) 0.019 Tumor recurrence    9 (17%)   3 (4%) 0.022 Multifocality   22 (41%)   23 (33%) 0.40 Number of I-131   1 (1-1)    1 (1-1) 0.083 treatments Total I-131 dose 100.4(76.2-105.4) 100.0 (30-103) 0.15 Dose/Treatment 100.0 (77.0-105.0) 100.0(100.0-104.5) 0.82 Total follow-up  10.5 (1-27)  14.0 (2-27) 0.82(months)*Eight cases had no information on tumor size;**Three cases had no information on tumor size;***Three cases had no information on the status of lymph nodemetastasis. If, on a conservative assumption, all these three cases hadno lymph node metastasis and were included in the analysis, theprevalence of lymph node metastasis in BRAF mutation-positive groupwould be 52% (instead of 55%) and the p value would be 0.0003 (insteadof <0.0001).⁺Five cases had no sufficient data to define tumor stage in the BRAFmutation-positive group. If, on a conservative assumption, all thesefive cases had a tumor stage less than III/IV and were included in theanalysis, the prevalence of tumor stages III/IV in BRAFmutation-positive group would be 30% (instead of 33%) and the p valuewould be 0.041 (instead of 0.019).

As shown in Table 1, in the present series of PTC, BRAF mutationoccurred most frequently in classic PTC (58%) and tall cell variant PTC(100%) and less frequently in follicular variant PTC (9%). Thisdifference is statistically highly significant (Table 3). A significantdifference in the high-risk pathologic features of extrathyroidalinvasion and neck lymph node metastasis as well as cancer recurrence wasalso seen among these different subtypes of PTC, correspondingly with ahigher occurrence in the classic and tall cell variant PTC and a muchlower occurrence in the follicular variant PTC (Table 3). We did notfind significant differences in the patient age and gender, tumormultifocality, and radioiodine treatments in these different tumorgroups in the present study. The difference in tumor size amongdifferent tumor subtype groups was significant apparently due to therelatively large size of the tumors in the follicular variant group andsmall size in the classic PTC group examined in the present study. TABLE3 Comparison of the clinicopathologic characteristics and BRAF mutationstatus in various subtypes of papillary thyroid cancers. Median(interquartile range) or N (%). Characteristic Classic Follicular TallCell P value N (total) 69 44 10 BRAF+ Mutation   40 (58%)    4 (9%)   10(100%) <.0001 Age at diagnosis  45.0 (38-57)  46.5 (33-55)  56.0 (40-76)0.18 Gender, male   25 (36%)   11 (25%)    2 (20%) 0.33 Tumor Size, cm 1.8 (1.1-2.7)*  2.9 (2.0-3.5)**  2.5 (2.0-5.5) 0.0013 Extrathyroidalinvasion   21 (30%)    1 (2%)    7 (70%) <.0001 Lymph node metastasis  30 (45%)***    5 (11%)    7 (70%) <.0001 Tumor stage 0.11 I   24 (38%)  13 (30%)    2 (20%) II   24 (38%)   25 (57%)    4 (40%) III   15 (23%)   4 (9%)    3 (30%) IV    1 (2%)    2 (5%)    1 (10%) Tumor stage,III/IV   16 (25%)⁺    6 (14%)    4 (40%) 0.13 Tumor recurrence    9(13%)    0 (0%)    3 (30%) 0.0024 Multifocality   30 (43%)   10 (23%)   5 (50%) 0.054 Number of I-131 treatments    1 (1-1)    1 (1-1)    1(1-1) 0.84 Total I-131 dose 100.0 (31.7-105.0) 100.0 (51.4-103.4) 105.0(51.0-105.0) 0.87 Dose/Treatment 100.0 (86.0-104.5) 100.0 (100.0-104.5)105.0 (78.0-127.5) 0.61 Total follow-up (months)  15.0 (2-27)  12.5(2-27)  6.0 (1-20) 0.58*Ten cases had no information on tumor size;**One cases had no information on tumor size;***Three cases had no information on the status of lymph nodemetastasis. If, on a conservative assumption, all these three cases hadno lymph node metastasis and were included in the analysis, theprevalence of lymph node metastasis in classic PTC would be 43% (insteadof 45%) and the p value would be 0.0012 (instead of <0.0001).⁺Five cases had no sufficient data to define tumor stage in the classicPTC group.BRAF Mutation is Inherently Associated with a Poor Prognosis of PTC

As patient age and gender, and tumor size and multifocality may allpotentially affect the clinical outcome and prognosis of thyroid cancer(18); (15), (17); (24) we next performed a multivariate analysis withthe adjustment for these factors on the correlation between the BRAFmutation status and each of the high-risk pathologic features (i.e.,tumor extrathyroidal invasion, neck lymph node metastasis, and advancedtumor stages) and tumor recurrence. For tumor recurrence, an adjustmentfor radioiodine treatment was also made since such treatment may alterthe clinical outcome of the cancer. As shown in Table 4, with such amultivariate analysis, a significant association (high odd ratio) ofBRAF mutation with each of the high-risk pathologic features and tumorrecurrence was still observed. Kaplan-Meier estimate revealed asignificantly lower tumor recurrence-free probability in PTC patientswith BRAF mutation (FIG. 2). Cox proportional hazards regressionanalysis on tumor recurrence, adjusting for age at diagnosis, gender,tumor size and multifocality, and total dose of I-131 resulted in astatistically significant relative risk of 37.68 (95% CI=1.17-1217.22,p=0.041) for tumor recurrence associated with BRAF mutation. Theseresults suggest that BRAF mutation plays an important role in thetumorigenesis of PTC and inherently predicts a poor prognosis for thesecancers. TABLE 4 Multivariate analysis on adjusted odds ratios for theassociation of BRAF mutation with clinicopathlogic outcomes of patientswith papillary thyroid cancer BRAF Mutation Odds Ratio 95% ConfidenceInterval P value Tumor Stage III/IV* 3.59 1.14-11.34 0.029 Lymph NodeMetastasis* 7.74 2.81-21.35 <.0001 Extrathyroidal Invasion* 8.002.80-22.85 0.0001 Tumor Recurrence⁺ 35.80  2.02-633.47 0.015*Adjusted for age at diagnosis, gender, multifocality, and tumor size⁺Also adjusted for total I-131 treatment

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1. A method for distinguishing malignant from benign thyroid samples,comprising: determining presence of a T→A transversion at nucleotide1796 of BRAF according to SEQ ID NO: 1 in a thyroid sample of a human,wherein presence of the transversion indicates a malignant thyroidneoplasm and absence of the transversion indicates a benign neoplasm orsample.
 2. The method of claim 1 wherein the thyroid sample is a fineneedle aspirate (FNA).
 3. The method of claim 1 wherein the thyroidsample is a tissue sample.
 4. The method of claim 1 wherein the thyroidsample is a cytological sample.
 5. The method of claim 1 furthercomprising: providing a diagnosis based on the presence or absence ofthe transversion.
 6. The method of claim 1 further comprising: providinga prognosis based on the presence or absence of the transversion.
 7. Themethod of claim 1 further comprising: determining a therapeutic regimenfor the human using as a factor the presence or absence of thetransversion.
 8. The method of claim 3 wherein the sample has afollicular morphology.
 9. The method of claim 3 wherein the sample as apapillary morphology.
 10. A method for distinguishing malignant frombenign thyroid samples, comprising: determining presence of a T→Atransversion at nucleotide 1796 of BRAF according to SEQ ID NO: 1 in ablood sample of a human, wherein presence of the transversion indicatesa malignant thyroid neoplasm in the human and absence of thetransversion indicates a benign neoplasm or no neoplasm.
 11. A methodfor detecting a mutation at nucleotide 1796 of BRAF, comprising:amplifying all or part of exon 15 of BRAF from a test sample to formamplified products, wherein said part comprises at least nucleotides1792 to 1799 of BRAF; digesting the amplified products with restrictionendonuclease TspRI to form digested products; identifying a mutation atnucleotide 1796 if the digested products contain: one fragment fewerthan digested products formed when using wild-type BRAF as a templatefor amplifying and digesting; or one additional fragment compared todigested products formed when using wild-type BRAF as a template foramplifying or digesting.
 12. The method of claim 11 wherein the testsample is from a thyroid.
 13. The method of claim 11 wherein the testsample is an FNA from a thyroid.
 14. The method of claim 11 wherein thetest sample is a tissue sample from a thyroid.
 15. A method of treatinga thyroid cancer patient, comprising: administering to the patient aneffective amount of an inhibitor of BRAF serine/threonine kinaseactivity or expression.
 16. The method of claim 15 wherein the inhibitoris an antibody which binds to BRAF serine/threonine kinase.
 17. Themethod of claim 15 wherein the inhibitor is an antisense oligonucleotidewhich is complementary to mRNA encoding BRAF serine/threonine kinase.18. The method of claim 15 wherein the inhibitor is siRNA which iscomplementary to mRNA encoding BRAF serine/threonine kinase.
 19. Themethod of claim 15 wherein the inhibitor is an antisense oligonucleotidewhich is made from an antisense construct.
 20. A method of treating athyroid cancer patient, comprising: administering to the patient aneffective amount of an inhibitor of Ras-Raf-MAPK pathway or Raf/MEK/ERKsignaling pathway.
 21. The method of claim 20 wherein the inhibitor isCI
 1040. 22. The method of claim 20 wherein the inhibitor is BAY43-9006.
 23. The method of claim 6 wherein the presence of thetransversion indicates a higher risk of neck lymph node metastasis. 24.The method of claim 6 wherein the presence of the transversion indicatesa higher risk of cancer recurrence.